(1) Field of the Invention
The present invention relates to a multiplex RT-PCR/PCR method, which enables in a single assay the simultaneous detection of any combination of bovine rotavirus, bovine coronavirus, Cryptosporidium parvum, and optionally, Escherichia coli strains producing K99 pili and heat-stable enterotoxin STa or STb.
(2) Description of Related Art
In the United States, bovine rotavirus, bovine coronavirus, Cryptosporidium parvum, and Escherichia coli strains producing K99 pili or fimbriae or heat-stable enterotoxin STa are the five most common infectious agents in neonatal calf diarrhea, commonly referred to as scours. The relative incidence of these pathogens in a multi-state survey of causes of scours in beef cattle was 35% for rotavirus and coronavirus combined, 24% for Cryptosporidium parvum, 22% for Escherichia coli, and 19% for others. The percent involvement in a study of scours in dairy calves was 46% for rotavirus, 15% for coronavirus, 23% for Cryptosporidium parvum, 13% for Salmonella spp., and 3% for Escherichia coli. Identification of these pathogens is a time consuming and laborious process.
For detecting bovine rotavirus and bovine coronavirus, electron microscopy or antigen-detection ELISA have been the standard detection method. Virus isolation is an attempt to isolate the virus in cell culture and then characterizing the isolated virus by standard virological methods. Other methods for detecting bovine rotavirus and bovine coronavirus include demonstrating the presence of rotavirus particles by negative staining electron microscopy, detecting rotavirus or coronavirus antigens by an antigen-detection enzyme linked immunosorbant assay (ELISA), or detection of the rotavirus or coronavirus RNA genome reverse transcription-polymerase chain reaction (RT-PCR) assays. For example, U.S. Pat. No. 5,959,093 to Saif et al. provides nucleic acid sequences encoding the VP4 and VP7 proteins from several bovine rotavirus serotypes and discloses a PCR method and primers for isolating cDNA encoding VP6.
Cryptosporidium parvum is detected by light microscopic examination of fecal smears for oocysts or by PCR of fecal samples using Cryptosporidium parvum specific oligonucleotide primers. For example, U.S. Pat. No. 5,770,368 to De Leon et al. discloses a method for detecting encysted forms of Cryptosporidium that are viable and infectious. The method involves isolating oocysts, inducing transcription of the heat shock protein (HSP) genes, and detecting the induced transcripts by RT-PCR. Alternatively, infectivity is determined by cultivating the Cryptosporidium on susceptible cells and either amplifying HSP DNA from infected cells by PCR or induce HSP transcription and detecting the induced transcripts by RT-PCR.
Escherichia coli producing K99 pili are detected by culturing fecal samples in specialized media and demonstrating the presence of K99 pili by immunological methods or by PCR using oligonucleotide primers specific to Escherichia coli strains producing K99 pili or heat-stable enterotoxin STa (Franck et al., J. Clin. Microbiol. 36: 1795-1797 (1998).
PCR is generally considered the most sensitive and rapid method for detecting nucleic acids of a pathogen in a particular sample. PCR is well known in the art and has been described in U.S. Pat. No. 4,683,195 to Mullis et al., U.S. Pat. No. 4,683,202 to Mullis, U.S. Pat. No. 5,298,392 to Atlas et al., and U.S. Pat. No. 5,437,990 to Burg et al. In the PCR step, oligonucleotide primer pairs for each of the target pathogens are provided wherein each primer pair comprises a first nucleotide sequence complementary to a sequence flanking the 5′ end of the target nucleic acid sequence and a second nucleotide sequence complementary to a nucleotide sequence flanking the 3′ end of the target nucleic acid sequence. The nucleotide sequences comprising each oligonucleotide primer pair are specific to particular pathogen to be detected and do not cross-react with other pathogens.
Multiple infectious agents are frequently involved in outbreaks of neonatal calf diarrhea. Thus, diarrhea in neonatal calves can be the result of infection by one or more of bovine rotavirus, bovine coronavirus, Cryptosporidium parvum, and Escherichia coli producing K99 pili or heat-stable enterotoxin STa. However, currently used assays have severe limitations in detecting these agents. Virus isolation and bacterial culture methods are labor intensive and costly and can involve days or weeks of culturing. Electron microscopy is also labor intensive and also is of limited sensitivity. ELISA is also a time consuming method. However, the primary drawback of ELISA is that it can result in false negatives when antigen-antibody complexes are shed instead of free virus particles and ELISA has limited sensitivity. None of the above methods are suitable for the simultaneous detection of multiple pathogens in a sample. PCR is a sensitive and rapid method for detecting pathogens, and it is amenable to simultaneously detecting multiple pathogens in a sample; however, using PCR for the simultaneous detection of multiple pathogens in a sample has been problematic. The primary obstacles to simultaneous detection of multiple pathogens have been cross-reactivity and preferential amplification of particular target sequences in the sample at the expense of the other target sequences in the sample. While U.S. Pat. No. 5,756,701 to Wu et al. discloses a multiplex PCR method for simultaneously detecting Salmonella spp., Yersinia spp., and Escherichia coli in a sample, the method is specific for the aforementioned bacterial species and does not include RT-PCR for detecting RNA viruses. U.S. Pat. No. 5,882,856 to Shuber also discloses a multiplex PCR method; however, the method uses chimeric primers comprising a sequence complementary to the target sequence covalently linked to a non-complementary sequence. Franck et al., J. Clin. Microbiol. 36: 1795-1797 (1998), discloses a multiplex PCR method for detecting particular Escherichia coli strains that encode K99 pili or heat-stable enterotoxin STa. However, the method does not include RT-PCR for detecting RNA viruses. In general, because of the difficulty in developing PCR methods, particularly RT-PCR methods, that enable simultaneous detection of multiple pathogens in a sample, most samples to be analyzed by PCR for multiple pathogens, are separately tested for each of the multiple pathogens in separate PCR reactions.
Because current methods for detecting the four most important infectious agents in neonatal calf diarrhea requires performing four separate assays, there is a need for a method which would enable the simultaneous detection of the four most important infectious agents involved in neonatal calf diarrhea. Simultaneous detection would provide substantial savings in cost and time in determining which of the four infectious agents is involved in a particular outbreak of neonatal calf diarrhea. Because simultaneous detection of any combination of these four agents in a single assay would avoid the potential for overlooking dual or even triple infections, the appropriate therapy could be initiated in a more timely and effective manner.